Therapeutic Nucleic Acids
Gene therapy is a major area of research in drug development. Gene therapy has been considered a desirable mechanism to correct genetic defects resulting in diseases associated with failure to produce certain proteins and to overcome acquired diseases such as autoimmune diseases and cancer. Gene therapy could provide a new prophylactic approach for the treatment of many diseases. A technological barrier to commercialization of gene therapy, however, is the need for practical, effective and safe means for polynucleotide delivery and sustained and/or controlled release. Polynucleotides do not readily permeate the cellular membrane due to the charge repulsion between the negatively charged membrane and the high negative charge on the polynucleotide. As a result, polynucleotides have poor bioavailability and uptake into cells, typically <1%. In animal models, viral-based vectors have been used successfully to administer genes to a desired tissue. In some cases, these approaches have led to long-term (>2 years) expression of therapeutic levels of the protein. However, the limitations of viral-based approaches have been extensively reported. For instance, re-administration is not possible with these vectors because of the humoral immune response generated against the viral proteins. In addition to manufacturing challenges to obtain adequate reproducible vector supply, there are also significant safety concerns associated with viral vectors, particularly for those targeting the liver for gene expression. Not withstanding the problems associated with viral gene therapy, viruses have been considered by many to be more efficient than non-viral delivery vehicles.
The silencing or down regulation of specific gene expression in a cell can be affected by oligonucleic acids using techniques known as antisense therapy, RNA interference (RNAi), and enzymatic nucleic acid molecules. Antisense therapy refers to the process of inactivating target DNA or mRNA sequences through the use of complementary DNA or RNA oligonucleic acids, thereby inhibiting gene transcription or translation. An antisense molecule can be single stranded, double stranded or triple helix. Other agents capable of inhibiting expression are for example enzymatic nucleic acid molecules such as DNAzymes and ribozymes, capable of specifically cleaving an mRNA transcript of interest. DNAzymes are single-stranded deoxyribonucleotides that are capable of cleaving both single- and double-stranded target sequences. Ribozymes are catalytic ribonucleic acid molecules that are increasingly being used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. RNA interference is a method of post-transcriptional inhibition of gene expression that is conserved throughout many eukaryotic organisms. It helps to control which genes are active and how active they are. Two types of small RNA molecules—microRNA (miRNA) and small interfering RNA (siRNA)—are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to specific other RNAs and either increase or decrease their activity, for example by preventing a messenger RNA from producing a protein. RNA interference has an important role in defending cells against parasitic genes—viruses and transposons—but also in directing development as well as gene expression in general. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. Development of RNAi-based therapeutics is relatively new to the pharmaceutical industry. Although many of the obstacles to the development of such drugs have been overcome, optimal delivery of the RNAi compounds to the appropriate tissues and into the cells is still a challenge.
Delivery of Nucleic Acids
A problem of non-viral gene therapy is to achieve the delivery and expression of sufficient nucleic acid to result in a tangible, physiologically relevant expression. Although DNA plasmids in isotonic saline (so-called “naked” DNA) were shown several years ago to transfect a variety of cells in vivo, such unprotected plasmids are susceptible to enzymatic degradation leading to irreproducibility in uptake and highly variable expression and biological responses in animal models. The very low bioavailability of “naked” plasmid in most tissues also requires high doses of plasmids to be administered to generate a pharmacological response. The field of non-viral gene delivery has therefore been directed to the development of more efficient synthetic delivery systems capable to increase the efficiency of plasmid delivery, confer prolonged expression and provide for storage stable formulations as is expected of other pharmaceutical formulations.
Chemical methods which facilitate the uptake of DNA by cells include the use of DEAE-Dextran. However this can result in loss of cell viability. Calcium phosphate is also a commonly used chemical agent which, when co-precipitated with DNA, introduces the DNA into cells.
Physical methods to introduce DNA have become effective means to reproducibly transfect cells. Direct microinjection is one such method which can deliver DNA directly to the nucleus of a cell (Capecchi 1980, Cell, 22, 479). This allows the analysis of single cell transfectants. So called “biolistic” methods physically insert DNA into cells and/or organelles using a high velocity particles coated with DNA. Electroporation is one of the most popular methods to transfect DNA. The method involves the use of a high voltage electrical charge to momentarily permeabilize cell membranes making them permeable to macromolecular complexes. However physical methods to introduce DNA do result in considerable loss of cell viability due to intracellular damage. More recently still a method termed immunoporation has become a recognized technique for the introduction of nucleic acid into cells, (Bildirici et al 2000, Nature, 405, 298). Transfection efficiency of between 40-50% is achievable depending on the nucleic acid used. These methods therefore require extensive optimization and also require expensive equipment.
To overcome the problem of degradation of nucleic acids, typically plasmid DNA (“pDNA”), or siRNAs/microRNA and enhance the efficiency of gene transfection, cationic condensing agents (such as polybrene, dendrimers, chitosan, lipids, and peptides) have been developed to protect the nucleic acids by condensing it through electrostatic interactions. However, the use of condensed plasmid particles for transfection of a large number of muscle cells in vivo, for example, has not been successful as compared to transfection of “naked” DNA.
Additional strategies that include the modulation of the plasmid surface charge and hydrophobicity by interaction with protective, interactive non-condensing systems have shown advantages over the use of “naked” DNA for direct administration to solid tissues (e.g., International Application Publication No. WO 96/21470).
Biodegradable microspheres that encapsulate the nucleic acid have also been used in gene delivery. For example, International Application Publication No. WO 00/78357 disclosed matrices, films, gels and hydrogels which include hyaluronic acid derivatized with a dihydrazide and crosslinked to a nucleic acid forming slow release microspheres.
Lipid based drug delivery systems are well known in the art of pharmaceutical science. Typically they are used to formulate drugs having poor bioavailability or high toxicity or both. Among the prevalent dosage forms that have gained acceptance are many different types of liposomes, including small unilamellar vesicles, multilamellar vesicles and many other types of liposomes; different types of emulsions, including water in oil emulsions, oil in water emulsions, water-in-oil-in-water double emulsions, submicron emulsions, microemulsions; micelles and many other hydrophobic drug carriers. These types of lipid based delivery systems can be highly specialized to permit targeted drug delivery or decreased toxicity or increased metabolic stability and the like. Extended release in the range of days, weeks and more are not profiles commonly associated with lipid based drug delivery systems in vivo. Liposomes that consist of amphiphilic cationic molecules are useful non-viral vectors for gene delivery in vitro and in vivo. In theory, the cationic head of the lipid associates with the negatively charged nucleic acid backbone of the DNA to form lipid:nucleic acid complexes. The lipid:nucleic acid complexes have several advantages as gene transfer vectors. Unlike viral vectors, the lipid:nucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they may evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency. The use of cationic lipids (e.g. liposomes) has become a common method since it does not have the degree of toxicity shown by chemical methods.
There are a number of publications that demonstrate convincingly that amphiphilic cationic lipids can mediate gene delivery in vivo and in vitro, by showing detectable expression of a reporter gene in culture cells in vitro. Because lipid:nucleic acid complexes are on occasion not as efficient as viral vectors for achieving successful gene transfer, much effort has been devoted in finding cationic lipids with increased transfection efficiency (Gao et al., 1995, Gene Therapy 2, 710-722).
Several works have reported the use of amphiphilic cationic lipid:nucleic acid complexes for in vivo transfection both in animals, and in humans (reviewed in Thierry et al., Proc. Natl. Acad. Sci. USA 1995, 92, 9742-9746). However, the technical problems for preparation of complexes having stable shelf-lives have not been addressed. For example, unlike viral vector preparations, lipid:nucleic acid complexes are unstable in terms of particle size. It is therefore difficult to obtain homogeneous lipid:nucleic acid complexes with a size distribution suitable for systemic injection. Most preparations of lipid:nucleic acid complexes are metastable. Consequently, these complexes typically must be used within a short period of time ranging from 30 minutes to a few hours. In clinical trials using cationic lipids as a carrier for DNA delivery, the two components were mixed at the bed-side and used immediately. The structural instability along with the loss of transfection activity of lipid:nucleic acid complex with time have been challenges for the future development of lipid-mediated gene therapy. Many of the recent developments in the field have focused on modification of the cationic system by combining a proven cationic delivery agent with another moiety. However, cationic backbone conjugates have not been successful in overcoming toxicity and none are approved for therapeutic use.
International Application Publication No. WO 95/24929 disclosed encapsulation or dispersion of genes in a biocompatible matrix, preferably biodegradable polymeric matrix, where the gene is able to diffuse out of the matrix over an extended period of time. Preferably the matrix is in the form of a microparticle such as a microsphere, microcapsule, a film, an implant, or a coating on a device such as a stent.
U.S. Pat. No. 6,048,551 disclosed a controlled release gene delivery system utilizing poly(lactide-co-glycolide) (PLGA), hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate, and the Ludragit R, L, and E series of polymers and copolymer microspheres to encapsulate the gene vector.
U.S. Application Publication No. 20070141134 discloses compositions that enhance the intracellular delivery of polynucleotides, wherein a polynucleotide can be incorporated into a PEG shielded micelle particle to facilitate the delivery of the polynucleotide across a cellular membrane. Incorporation of the polynucleotide into the shielded micelle particle is provided by covalent and non-covalent means. Other cell targeting agents may also be covalently coupled to the shielded micelle particle to enhance localization in the body.
International Patent Application Publication No. WO 2008/124634 discloses a method for encapsulating nucleic acids, particularly siRNAs, shRNAs, microRNAs, gene therapy plasmids, and other oligonucleotides in biodegradable polymer, whereby the nucleic acids are formulated into reverse micelles composed of non-toxic and/or naturally-occurring lipids prior to nanoparticle formation by nanoprecipitation.
International Application Publication No. WO 2009/127060 discloses a nucleic acid-lipid particle, comprising, in addition to the nucleic acid, a cationic lipid, a non-cationic lipid and a conjugated lipid that inhibits aggregation of the particles.
International Patent Application Publication No. WO 2010/007623 to some inventors of the present invention, published after the priority date of the present invention, discloses compositions for extended release of hydrophobic molecules such as steroids and antibiotics, comprising a lipid-based matrix comprising a biodegradable polymer.
Ideally sustained release drug delivery systems should exhibit kinetic and other characteristics readily controlled by the types and ratios of the specific excipients used. There remain an unmet need for improved nucleic acid compositions and methods for controlled and extended delivery of therapeutic nucleic acid agents to appropriate tissues and into cells for gene therapy. Nowhere in the prior art it was suggested that matrix compositions comprising lipids and biocompatible polymer will possess improved properties for delivering nucleic acid based agents.